Autosomal Allelic Inactivation: Variable Replication and Dosage Sensitivity

Altered Replication Timing of IGH Alleles in Burkitt's Lymphoma Depends on IGH Breakpoint Position.

Modified allelic replication in lymphocytes of patients with neurofibromatosis type 1

Besides genome-wide patterns of replication timing (RT), some genes display allelic replication asynchrony in stem cells, brought about by stochastic events and genetic polymorphisms. Whether epigenetic modifications control asynchronous replication remains unclear. Here, we explore domains controlled by genomic imprinting, where parental DNA methylation imprints mediate allele-specific gene expression. Our genome-wide and locus-specific assays in mono-parental and hybrid mouse ESCs reveal pronounced RT asynchrony-which is parent-of-origin dependent and lost upon neural differentiation-at the Dlk1-Dio3 and Snrpn domains, which both comprise lncRNA polycistrons. Generating a range of mutant lines, we find that asynchronous replication at Dlk1-Dio3 is mediated by differential DNA methylation, and that the lncRNA Meg3 controls early replication across parts of the domain on the maternal chromosome. Moreover, we find no evidence that RT and organisation into TADs are linked in this domain. The combined replication timing, DNA methylation, 3D chromatin structure, and gene expression data highlight how parental methylation imprints and lncRNA expression control replication and can override RT domain organisation.

Asynchronous DNA replication is an epigenetically determined feature found in all cases of monoallelic expression, including genomic imprinting, X-inactivation, and random monoallelic expression of autosomal genes such as immunoglobulins and olfactory receptor genes. Most genes of the latter class were identified in experiments focused on genes functioning in the chemosensory and immune systems. We performed an unbiased survey of asynchronous replication in the mouse genome, excluding known asynchronously replicated genes. Fully 10% (eight of 80) of the genes tested exhibited asynchronous replication. A common feature of the newly identified asynchronously replicated areas is their proximity to areas of tandem gene duplication. Testing of other clustered areas supported the idea that such regions are enriched with asynchronously replicated genes.

Several unique features distinguish imprinted from nonimprinted genes, including the unusual replication behavior the unequal methylation and the differential expression of imprinted alleles [1]. The replication timing in S phase of the two homologous alleles of a normal, nonimprinted gene is highly synchronous [2, 3]. Housekeeping genes replicate early, constitutive heterochromatic regions replicate late and tissue-specific genes replicate earlier when they are expressed than when they are not [2-4]. In contrast, imprinted genes which, by definition, display allele-specific expression replicate asynchronously [2-5].The relative order of replication of homologous alleles as well as that of different loci can be elegantly compared with fluorescence in situ hybridization (FISH) on interphase nuclei [2-5]. Unreplicated DNA segments give singlet hybridization signals in normal diploid cells, while replicated loci are characterized by doublets. The distribution of these two patterns can be used to determine the S phase replication time of any DNA sequence. Moreover, determination of the singlet/doublet ratio allows a good estimation of the degree of replication asynchrony of two homologous alleles [2-5].Using cell lines with deletions, disomies or associated FISH-detectable centromeric satellite polymorphisms, Kitsberg et al. [4] found that the paternal allele was the early replicating one in all the imprinted genes which they had analyzed. Subsequently, however, Knoll et al. [5] detected genes in the imprinted Prader-Willi region on chromosome 15, which also displayed other patterns. Therefore, it seems necessary to specify the relative timing of maternally and paternally derived alleles for each individual asynchronously replicating gene. Unfortunately, this is so far only feasible with a very restricted number of sequences.

Analysis of a cell’s replication timing (RT) provides insight into how genes replicate, early or late, during the S-phase of the cell cycle. RT is cell-type specific, inheritable, and has been correlated to gene expression in normal and diseased states. However, most studies have been limited to somatic cells. Very little is known about RT control in early mouse embryos, and how it correlates with the start of transcription during zygote gene activation (ZGA), at the 2-cell stage. In this study, we develop an in-house single-cell multiomics approach to simultaneously analyze RT and gene expression in individual cells of the mouse 1-cell, 2-cell, and 4-cell embryos. We detect that RT is established at the 1-cell stage prior to ZGA. Surprisingly, we observe that the coordinated RT and gene expression control is different in early totipotent embryos, compared to previously published studies in somatic cells. Late replicating regions correlate with higher gene expression and open chromatin in the early developing embryos. Lastly, we perform an integrated pseudo time trajectory analysis combining RT and gene expression information per cell.

Replication timing (RT) allows us to analyze temporal patterns of genome-wide replication, that is, if genes replicate early or late during the S-phase of the cell cycle. RT has been linked to gene expression in normal and diseased acute and chronic states, such as cancer. However, studies done to date focused on bulk cell populations that required tens of thousands of cells for RT analysis. Here, we developed an affordable novel single-cell (sc) multiomics approach to simultaneously analyze RT and gene expression from cells or nuclei. We used this approach to generate single-cell RT profiles and sc-gene expression data from the well-established human liver cancer cell line, HepG2. We demonstrated that as few as 17 mid S-phase cells were sufficient to produce cell type–specific pseudo bulk RT profiles that had a high correlation to previously published HepG2 bulk RT profiles. The single-cell RT profiles allowed us to visualize how individual cells progressed through genome replication. We were also able to demonstrate cell-specific correlations between RT and gene expression, which to our knowledge, has not been reported. We observed trends that were conserved between individual cells, as well as cell-to-cell variations, which were not possible to detect with the bulk RT studies.

Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.

Primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) are associated chronic inflammatory diseases with malignant potential. Loss of replication synchrony during the S-phase of the cell cycle has been shown to be linked to several malignant and premalignant states. This study evaluated temporal differences in replication timing between these diseases. The replication pattern of peripheral blood lymphocytes obtained from patients with PSC and IBD and healthy individuals was analyzed by fluorescence in situ hybridization (FISH) in 2 pairs of alleles, in 15qter and 13qter. Asynchrony was determined by the presence of 1 single and 1 set of double dots in the same cell. Samples from subjects with PSC showed significantly greater temporal differences in replication timing, in contrast to the high level of synchrony observed in samples from healthy individuals (p = 0.045). Samples from IBD patients exhibited a nonsignificant increase in replication asynchrony. We believe that these results reflect impairment in the replication control of structural homologous loci in PSC, and that this phenomenon may be correlated with the inflammation-induced malignant potential of this condition.

A major activity of a pre-B lymphocyte is the stepwise rearrangement of immunoglobulin heavy and light chain genes. When a pre-B cell has successfully completed the process of rearrangement at both heavy and light chain loci, this dividing lymphocyte undergoes a cellular transition into a mature B cell whereby it stops dividing and expresses membrane immunoglobulin tetramers (μm2L2) on its surface which will function as the antigen receptor. The process of rearrangement is of central importance to the generation of immunological diversity. However, it is imperative that in any given lymphocyte only a single heavy chain allele be productively rearranged (allelic exclusion), and that only a single κ or λ light chain allele be correctly rearranged (allelic and isotypic exclusion). These processes of allelic and isotypic exclusion are unique to the immune system and ensure that any lymphoid clone expresses only a single antigen receptor.

Rapid Irreversible Transcriptional Reprogramming in Human Stem Cells Accompanied by Discordance between Replication Timing and Chromatin Compartment.

Epigenome The temporal order of DNA replication is conserved from yeast to humans, but its biological significance remains unclear. Klein et al. eliminated the protein RIF1, a master regulator of replication timing, in several human cell lines. RIF1 loss during the G1 phase of the cell cycle resulted in a heterogeneous, nearly random replication timing program from the first S phase that persisted even in stable RIF1-null clones. Altered replication timing was followed by replication-dependent redistribution of active and repressive histone modifications and alterations in genome architecture. These results support a model in which replication timing orchestrates the epigenetic state of newly replicated chromatin. Science , this issue p. [371][1] [1]: /lookup/doi/10.1126/science.aba5545

Choreography of Ig allelic exclusion

Allelic exclusion in kappa light-chain synthesis is thought to result from a feedback mechanism by which the expression of a functional kappa light chain on the surface of the B cell leads to an intracellular signal that down-regulates the V(D)J recombinase, thus precluding rearrangement of the other allele. Whereas such a feedback mechanism clearly plays a role in the maintenance of allelic exclusion, here we provide evidence suggesting that the initial establishment of allelic exclusion involves differential availability of the two kappa alleles for rearrangement. Analysis of kappa+ B-cell populations and of individual kappa+ B cells that have rearranged only one allele demonstrates that in these cells, critical sites on the rearranged allele are unmethylated, whereas the nonrearranged allele remains methylated. This pattern is apparently generated by demethylation that is initiated at the small pre-B cell stage, on a single allele, in a process that occurs prior to rearrangement and requires the presence in cis of both the intronic and 3' kappa enhancers. Taken together with data demonstrating that undermethylation is required for rearrangement, these results indicate that demethylation may actually underly the process of allelic exclusion by directing the initial choice of a single kappa allele for rearrangement.

Alterations of H19 Imprinting and IGF2 Replication Timing Are Infrequent in Beckwith–Wiedemann Syndrome